Many peptides and proteins can be produced via recombinant means in a variety of expression systems, e.g., various strains of bacterial, fungal, mammalian or insect cells. However, when bacteria are used as host cells for heterologous gene expression, several problems frequently occur.
For example, heterologous genes encoding small peptides are often poorly expressed in bacteria. Because of their size, most small peptides are unable to adopt stable, soluble conformations and are subject to intracellular degradation by proteases and peptidases present in the host cell. Those small peptides which do manage to accumulate when directly expressed in E. coli or other bacterial hosts are usually found in the insoluble or "inclusion body" fraction, an occurrence which renders them almost useless for screening purposes in biological or biochemical assays.
Moreover, even if small peptides are not produced in inclusion bodies, the production of small peptides by recombinant means as candidates for new drugs or enzyme inhibitors encounters further problems. Even small linear peptides can adopt an enormous number of potential structures due to their degrees of conformational freedom. Thus a small peptide can have the `desired` amino-acid sequence and yet have very low activity in an assay because the `active` peptide conformation is only one of the many alternative structures adopted in free solution. This presents another difficulty encountered in producing small heterologous peptides recombinantly for effective research and therapeutic use.
Inclusion body formation is also frequently observed when the genes for heterologous proteins are expressed in bacterial cells. These inclusion bodies usually require further manipulations in order to solubilize and refold the heterologous protein, with conditions determined empirically and with uncertainty in each case.
If these additional procedures are not successful, little to no protein retaining bioactivity can be recovered from the host cells. Moreover, these additional processes are often technically difficult and prohibitively expensive for practical production of recombinant proteins for therapeutic, diagnostic or other research uses.
To overcome these problems, the art has employed certain peptides or proteins as fusion "partners" with a desired heterologous peptide or protein to enable the recombinant expression and/or secretion of small peptides or larger proteins as fusion proteins in bacterial expression systems. Among such fusion partners are included lacZ and trpE fusion proteins, maltose-binding protein fusions, and glutathione-S-transferase fusion proteins [See, generally, Current Protocols in Molecular Biology, Vol. 2, suppl. 10, publ. John Wiley and Sons, New York, N.Y., pp. 16.4.1-16.8.1 (1990); and Smith et al, Gene, 67:31-40 (1988)]. As another example, U.S. Pat. No. 4,801,536 describes the fusion of a bacterial flagellin protein to a desired protein to enable the production of a heterologous gene in a bacterial cell and its secretion into the culture medium as a fusion protein.
However, often fusions of desired peptides or proteins to other proteins (i.e., as fusion partners) at the amino- or carboxyl- termini of these fusion partner proteins have other potential disadvantages. Experience in E. coli has shown that a crucial factor in obtaining high levels of gene expression is the efficiency of translational initiation. Translational initiation in E. coli is very sensitive to the nucleotide sequence surrounding the initiating methionine codon of the desired heterologous peptide or protein sequence, although the rules governing this phenomenon are not clear. For this reason, fusions of sequences at the amino-terminus of many fusion partner proteins affects expression levels in an unpredictable manner. In addition there are numerous amino- and carboxy-peptidases in E. coli which degrade amino- or carboxyl-terminal peptide extensions to fusion partner proteins so that a number of the known fusion partners have a low success rate for producing stable fusion proteins.
The purification of proteins produced by recombinant expression systems is often a serious challenge. There is a continuing requirement for new and easier methods to produce homogeneous preparations of recombinant proteins, and yet a number of the fusion partners currently used in the art possess no inherent properties that would facilitate the purification process. Therefore, in the art of recombinant expression systems, there remains a need for new compositions and processes for the production and purification of stable, soluble peptides and proteins for use in research, diagnostic and therapeutic applications.